This invention relates to drugs in which peptides having affinity specific for phospholipids, preferably those which are contained in the constituents of lipid bilayers forming the surface layers of cells and of which the proportion in the outer part of each lipid bilayer increases when the cell is not normal, for example, in the case where it is damaged, denatured or activated, and biologically active substances bind to each other, deoxyribonucleic acid (DNA) which codes for the amino acid sequence of the drug in the case where the biologically active substance is a peptide, and processes for producing such drugs.
The invention also relates to a novel peptide having affinity specific for a phospholipid, preferably one which is contained in the constituents of lipid bilayers forming the surface layers of cells and of which the proportion in the outer part of each lipid bilayer increases when the cell is not normal, for example, in the case where it is damaged, denatured or activated, more preferably phosphatidylserine or phosphatidylethanolamine, further preferably phosphatidylserine, and DNA coding for the peptide.
The drugs and novel peptides of the invention are useful as preventives and therapeutics of diseases involving coagulopathy, inflammations and immune response.
Active studies are conducted today in connection with the creation of new drugs of high utility and as one of such studies an attempt is known that is directed to enhance the proportion in which a drug administered in vivo is delivered to the site for effective action while reducing the amount in which it becomes ineffective. This is an attempt at delivery of the drug in a site-specific manner, or an attempt of the so-called xe2x80x9ctargetingxe2x80x9d. A representative method of targeting is one that utilizes an antigen-antibody reaction. A method that may be mentioned as an example of this approach is one in which an F(abxe2x80x2)2 fragment of a tumor cell specific antibody (21B2) is bound to liposomes containing adriamycin (ADM) to prepare immunoliposomes such that ADM is delivered on tumor cells [I. Uyama et al., Jpn. J. Cancer Res., Vol. 85, 434 (1994)]. Another representative method of targeting is one that utilizes the interaction between a polypeptide-composed receptor and its ligand. A method that may be mentioned as an example of this approach is one in which an RGD polypeptide sequence having an ability to bind to a GPIIb/GPIIIa receptor is attached to the C terminus of phospholipase A2 (PLA2), whereby the PLA2 is delivered on the membrane surface of platelets (A.C.A.P.A. Bekkers et al., Thrombosis and Haemostasis, Vol. 74, 1138 (1995)). Other examples that have so far been reported include one in which a peptide having affinity for heparin present on the surface of cells is bound to superoxide dismutase (SOD) or complement regulatory proteins such that the SOD is delivered on the cell surface (M. Inoue et al., J. Biol. Chem., Vol. 266, 16409 (1991)) or the complement regulatory proteins is delivered on the cell surface (International Patent Publication WO/96/34965).
Peptides are known which themselves have no biological activity but which have selective affinity to the target site, whereby the binding of a biologically active substances to the target site of action is inhibited through a competitive reaction. An example of such peptides is a polypeptide that binds to a receptor on the surface of cells selectively, thereby inhibiting a ligand from binding to the receptor. An example of such polypeptides is a polypeptide fragment derived from the antigen recognizing site (or complementary determining region: CDR) of an anti-TNFxcex1 monoclonal antibody, which inhibits TNFxcex1 from binding to a receptor [E. Doring et al., Molecular Immunology, Vol. 31, 1059 (1994)]. Another example is a polypeptide which, by selective binding to a phospholipid, phosphatidylserine, inhibits a factor involved in the progress of a blood coagulation from binding to the phospholipid and which is composed of 30 amino acids at the terminus of the C2 region of a human factor VIII (International Patent Publication WO/90/15615) or 12 amino acids derived from the CDR of a phosphatidylserine recognizing antibody (Japanese Patent Public Disclosure KOKAI No. 92992/1993).
Another example that concerns the creation of new drugs of high utility is an attempt at imparting a new biologically active function to an already biologically active peptide by genetic engineering techniques. As regards TM which is known to suppress a blood clotting reaction, reports have been made of a TM derivative which has a fibrinolytic enzyme such as a tissue plasminogen activator (tPA) bound to a TM peptide (Japanese Patent Domestic Announcement KOHYO 505554/1992) and a TM derivative which has a specified amino acid sequence bound to the C terminus of a TM peptide so as to impart its action in enhancing the activity of antithrombin III and suppressing platelet aggregation (Japanese Patent Public Disclosure KOKAI No. 279497/1994).
There are known many drugs today that must be used in limited doses because of the side effects they have. For example, heparin and antithrombin III are used as anticoagulants but the use of these substances must occasionally be limited since they have side effects such as the tendency to cause bleeding.
When administering drugs, it is generally required to administer more than a certain dose in order to attain a specified efficacy and side effects to the patient are sometimes a serious problem. In addition, it is by no means rare that administering high doses of drugs will eventually often increase the economic burden on the part of the patient. Therefore, it is desired to develop drugs that have high enough activity to exhibit satisfactory efficacy in low doses such that the side effects from drug administration can be reduced and which can be used more extensively without increasing the economic burden on the patient.
Under the circumstances, the present inventors conducted intensive studies with a view to attaining the stated object. As a result, they found that a drug having affinity for a specified phospholipid that was created by binding a biologically active substance to a substance having affinity for the specified phospholipid had an improved ability to localize on the specified phospholipid, thereby achieving a marked enhancement of its action and efficacy. The inventors further found that this marked enhancement of the action and efficacy of the drug was dependent on the specified phospholipid, which led to the accomplishment of the present invention.
Thus, a first aspect of the present invention is a drug that contains both a substance having affinity for a phospholipid and a biologically active substance. The drug is preferably one having a novel substance that possesses both a portion having affinity for a desired phospholipid and a portion having biologically activity; since the drug is obtained as a different form than it inherently occurs in nature, it is a drug having a chimeric substance or a substance produced by fusion of different proteins. Herein, said affinity for a phospholipid or said portion having affinity for a phospholipid preferably originates from the substance having affinity for a phospholipid, whereas the biological activity or the portion having biological activity preferably originates from the biologically active substance. Specifically, the drug is one that contains both a substance having affinity for a specified phospholipid, preferably phosphatidylserine or phosphatidylethanolamine, more preferably phosphatidylserine, and a biologically active substance. Herein, the substance having affinity for a specified phospholipid is preferably a peptide or a peptide-containing substance. Said peptide has a sequence represented by the following general formula; preferably, it is composed of said sequence. It should be noted that all of the sequences to be described in this specification permit substitution, deletion, addition, insertion and so forth in part of their constituent elements unless their characteristics are impaired. Specifically, if the sequence is an amino acid sequence, one or more amino acids may be substituted, deleted, added, inserted or otherwise modified without impairing the activity of the sequence.
(A1)n1xe2x88x92(A2)n2xe2x88x92(A3)n3
(provided that in the general formula set forth above, A1 represents the amino acid sequence denoted by SEQ ID NO:1 or 10; A2 and A3 represent the amino acid sequences denoted by SEQ ID NOS:2 and 3, respectively; n1, n2 and n3 which represent the repeating numbers of A1, A2 and A3, respectively, are 0-5, 1-5 and 0-5 respectively; preferably, n1 is 0 or 1, n2 is 1, 2 or 3, and n3 is 0 or 1).
More preferably, the substance having affinity for a phospholipid has at least either of the following sequences:
(A1)1xe2x88x92(A2)1xe2x88x92(A3)1 (SEQ ID NO:8 or 9)
(A2)1xe2x88x92(A3)1 (SEQ ID NO:5)
(A2)2xe2x88x92(A3)1 (SEQ ID NO:6)
(A2)3xe2x88x92(A3)1 (SEQ ID NO:7)
(A2)2 (SEQ ID NO:22)
The drug according to the first aspect of the present invention preferably contains a peptide as the biologically active substance; specifically, at least one member selected from the group consisting of a factor involved in a blood coagulation system, a factor involved in a fibrinolytic system, a factor involved in an immune response reaction, a factor suppressing cytopathy, a factor inhibiting the activity of proteases and modified versions thereof, more preferably, at least one member selected from the group consisting of a factor suppressing a blood coagulation, a factor enhancing a fibrinolytic system, a factor suppressing a complement activating reaction, a factor suppressing the cytopathy due to active oxygen, a factor inhibiting the activity of proteases and modified versions thereof, further preferably at least one member selected from the group consisting of TM, the second region of UTI, MCP, UTI and modified versions thereof, particularly preferably a peptide having the amino acid sequence denoted by SEQ ID NO:4 or either of SEQ ID NOS: 23-25.
In a mode of containing both the biologically active substance and the substance having affinity for a specified phospholipid, the drug according to the first aspect of the invention preferably contains a linkage via a peptide bond, more preferably a linkage in which the N terminal amino acid of the substance having affinity for a phospholipid and the C terminal amino acid of the biologically active substance or, alternatively, the C terminal amino acid of the substance having affinity for a phospholipid and the N terminal amino acid of the biologically active substance are associated by a peptide bond, further preferably, a mode in which the N terminal amino acid of the substance having affinity for a phospholipid and the C terminal amino acid of the biologically active substance are associated by a peptide bond.
In its second aspect, the present invention provides a peptide having the amino acid sequence represented by the following general formula:
(A2)n2xe2x88x92(A3)n3
provided that A2 and A3 are the amino acid sequences denoted by SEQ ID NOS:2 and 3, respectively; n2 which is the repeating number of A2 is 2 or 3; n3 which is the repeating sequence of A3 is 0 or 1.
More preferably, the amino acid sequence of said peptide has either of the following sequences:
(A2)2xe2x88x92(A3)1 (SEQ ID NO:6)
(A2)3xe2x88x92(A3)1 (SEQ ID NO:7)
(A2)2 (SEQ ID NO:22)
According to its third aspect, the present invention provides DNA coding for the amino acid sequence of the drug according to the first aspect of the present invention or the peptide portion of said drug.
According to its fourth aspect, the present invention provides DNA coding for the amino acid sequence of the peptide according to the second aspect of the invention.
According to its fifth aspect, the present invention provides a process for producing the drug according to the first aspect of the invention.